Field of the Invention
The invention relates to a storage-stable lysis buffer mixture for extraction of nucleic acids from biological, preferably diagnostic samples. It is preferably connected with an extraction control.
Fields of application are molecular biology diagnostics, research, medical practice, gene-based analysis of biotechnological, agricultural and foodstuff products as well as criminal science.
Background of the Related Art
A large number of customary lysis buffers for extraction of nucleic acids contain chaotropic ion mixtures as salts. The pertinent methods are based on a method developed and first described by Vogelstein and Gillespie (Proc. Natl. Acad. Sci. USA, 1979, 76, 615-619) for preparative and analytical cleansing of DNA fragments from agarose gels. The method combines the dissolution of the agarose containing the DNA bands to be isolated in a saturated solution of a chaotropic salt (NaJ) with a binding of the DNA to glass particles.
A method for isolation of nucleic acids practicable for a large number of varying applications has been shown in U.S. Pat. No. 5,234,809 (Boom). There a method for isolation of nucleic acids from initial substances containing nucleic acid by incubation of the initial substance with a chaotropic lysis buffer and a solid phase binding the DNA is described.
The chaotropic lysis buffers implement both the lysis of the initial substance as well as the binding of the nucleic acids to the solid phase.
However, lysis buffers of this kind also denaturate enzymes necessary for the lysis of a biological sample in a short period of time. This is why customary systems require a separate addition of the individual components. So the lysis buffer is only put together during the lysis procedure and the individual components are added in succession in accordance with a certain order.
The sequence of extraction and detection can be checked via various forms of control: the extraction control to determine the quality of the extraction and a reaction control to determine the quality of the detection method.
From patent application DE 19840531, solutions for stabilised process controls are known, although they are limited to stabilised (RT) qPCR controls in the corresponding reaction vessels. They are applied to the vessel surfaces in question free of water. In this way, an examination of the reverse transcriptase reaction and an examination of the qPCR reaction are made possible. However, the extraction remains uncontrolled in this context.
In the case of the extraction of very slight copy counts of nucleic acids, so-called carrier nucleic acids are often used, as slight quantities of nucleic acids are instable in an aqueous solution. These carrier nucleic acids may not have any kind of homology to the extracted nucleic acids and must normally be added separately in nucleic acid extraction systems. Typical carrier nucleic acids are salmon sperm DNA, herring sperm DNA, yeast t RNA and polyadenyl RNA.
In conventional methods, the carrier nucleic acids are customarily added to the sample in the extraction. This demands an additional pipetting step. The extraction control is also added to the sample in a known quantity in the extraction. This demands a further additional pipetting step. This results in difficulties in storing the control stably in a liquid form, to the extent that it comprises pure nucleic acids. Nucleic acids in low concentrations tend to degrade.
After this, the extraction control is measured, following the reaction, via a detection method, and the recovery rate can determined on the basis of the loss of control nucleic acid.
To counteract the effect of instability and also to simulate the extraction process, phage particles or nucleic acids packed in phage particles are sometimes used. However, production of such controls is very time-consuming and thus expensive.
The lytic enzymes are added to the reaction as liquid components in a further pipetting step. The reaction control for the detection methods is added to the detection reaction separately, which for its part demands an additional pipetting step, or the mixture for the detection reaction itself contains a so-called amplification control.
The following disadvantages result from this for the user:
All the components must be put together in various pipetting steps, which leads to additional work and the risk of contaminations.
The components of such a system have to be stored at differing temperatures. All told, automation of such a system proves to be complicated.
A different approach is shown in WO 0034463. Here, there is a description of a lysis buffer which is not based on chaotropic salts and suitable detergents.
As WO 0034463 shows, such mixtures are also storage-stable for some time as solid formulations. In WO 03040386, the possibility of the use of such a system for standardised nucleic acid extraction and for standardised detection of nucleic acid is described.
Publication WO 03040386 describes reaction areas which have the potential for efficient and fast methods. However, the manufacture of matching products is very time-consuming and bound to the later reaction area. The manufacture of the reaction areas in method WO 03040386 is via freeze-drying.
The reaction areas are provided with the various components of the reaction system in a water-free or practically water-free condition. This is connected with a lot of time being needed for weighing and taking the components to the reaction area and prevents automation to a great extent.
The carrier nucleic acids are put into the reaction area for the lysis buffer via the lysis vessel, often together with the extraction control. This is done via so-called coating methods, in which a solution of the nucleic acid mixture is dried onto the wall of the reaction vessel.
In order to complete the reaction mix for the lysis procedure, all that is now needed is a so-called lysis buffer mixture.
The lysis buffer mixture contains non-chaotropic salts and, if necessary, detergents and is prepared as a liquid mixture. This mixture is then dried. After drying, lytic enzymes in a solid form are added and the resulting powder mixed very thoroughly. The powder is then weighed and distributed to the coated reaction areas. In this way, there is a stable mixture for the lysis of samples containing nucleic acid in the reaction areas, this mixture being stable at room temperature for six months.
Separate addition of the enzymes as a dry substance is indispensable in this method, as the enzymes would partly denaturate in the mixture used during the drying.
The extraction control is also used as a reaction control for the detection method.
This method provides the following advantages for the user:
The sample can be added in a liquid form, no further pipetting steps are necessary and an enlargement of the volume of liquid samples is possible.
But the method also has disadvantages, which are in the manufacture and the shelf life:
Manufacture of the aforementioned system is very time-consuming, which makes the system expensive. The system merely has a shelf life of six months, in which context the coated vessels must be stored at −20° C., the coated vessels filled with lysis buffer mixture can then be stored at room temperature for six months.
To sum up, it can be stated that the user has a number of advantages in this procedure, but the manufacturing process becomes more complicated, with the result that the system is made more expensive in the long run.